Valery I. Shestopalov, Ph.D.
Neurobiology of Retinal Disease, Glia-neuron Crosstalk in Neurodegeneration, Ocular Surface Microbiome
Mechanisms of Ganglion Cell Loss in Glaucoma, Ischemia and Optic Neuropathies. Microbial Ecology of the Ocular Surface
The Shestopalov Lab seeks to cure eye pathologies through deep understanding of the molecular and cellular mechanisms underlying disease initiation and progression. Our research is broadly focused on ocular pathologies leading to ganglion cell degeneration in the retina. Experimental evidence shows that complex network of interactions between glial cells, neurons and inflammatory cells in the retina become severely affected by injuries, ischemic and inflammatory pathologies. We explore the paradigm that therapeutic restoration of homeostatic signaling networks suppresses damage from injuries and facilitates recovery in cells and tissues affected by pathological factors. We detect affected pathways using high throughput molecular approaches, functional genomics and bioinformatics. This new knowledge is critically important in designing new treatments: key regulators of such pathways, identified by the analysis, are tested as potential drug targets.
Our translational projects study the role of microbial community in ocular surface infections. Our NIH ARRA-funded research efforts are part of the Human Microbiome Roadmap Project (HMP) and represent collaborative effort of basic and clinician scientists at UMMS Bascom Palmer Eye Institute and the Department of Ophthalmology at the University of Washington (Seattle, WA).
Systems approach in studies of ocular pathologies
Neuronal injury and loss in degenerative diseases of optic nerve and retina are facilitated by a combination of environmental stress, genetic factors and altered crosstalk between neurons and glia. This altered crosstalk facilitates glial toxicity, the area a renewed interest in the field. Our success in profiling the glia- and neuron-specific changes [1-3] has encouraged network analysis aimed at reconstructing the network of the glia-neuron cellular interactions. The in silico analysis of transcriptomic, proteomics, and epigenetic data using cutting edge informatics allowed us identify and prioritize the key regulators of pathological changes affecting optic nerve astrocytes in glaucoma .
Transcription factor NF-kappaB that controls inflammatory responses and was among the pathways most affected in glaucomatous astrocytes, has been selected as potential therapeutic target. We utilized transgenic GFAP-IκBα-DN mice possessing suppressed NF-κB in astrocytes (collaboration with Dr. JR Bethea), to test the hypothesis that such suppression is neuroprotective. As an excellent proof that our hypothesis is correct, these animals showed dramatically increased neuronal resistance in animal disease models of glaucoma, as well as optic neuritis and retinal ischemia-reperfusion [5, 6].
Combined, our results suggested that NF-κB is a master regulator of glia-induced inflammation and neurotoxicity in various ocular pathologies associated with axonal injury. This also validates our approach of systems-level analysis of complex retinal diseases, the project funded by R21 grant from NIH NEI.
The role of pannexins in neurodegenerative diseases of the eye
In this project we focus our scientific enquiry on the role pannexin1 (Panx1) protein in stem cell differentiation and degenerative diseases of the retina and optic nerve.
Pannexins are the newly discovered membrane protein structurally and evolutionary related to invertebrate gap junction proteins. Panx1 form transmembrane channels incapable of coupling into gap junctions, which distincts them from connexins that can form both gap junction channels and hemichannels. We have demonstrated that Panx1 and Panx2 are abundantly expressed in the ER and plasma membranes of lens and blood endothelial cells, skeletal and heart muscles, retinal ganglion cells and horizontal cells in the retina, and in the cochlea [1-4]. Physiological properties of the Panx1 channels allow the passage of ions, metabolites and small molecules including Ca2+, ATP, GSH, glutamate, arachidonic acid, etc. through by-lipid layers of plasma membrane and endoplasmic reticulum. While most often implicated in paracrine signaling, normal physiological function of Panx1 and a role in development remain poorly understood.
Significantly, the channel opening by ischemia, nitric oxide, extracellular ATP and glutamate has been suggested to facilitate neurotoxicity. The channel opening can, therefore, provide a pathway linking extracellular stressors or danger signals and intracellular toxicity pathways. Activation of such pathways in retinal ischemia, glaucoma and traumatic injury is known to facilitate neurodegeneration.
We have developed Panx1-specific antibodies and Panx1 conditional knockout (CKO) mouse model and were first to demonstrate the crucial role this channel plays in retinal ischemia-reperfusion injury. Using these new tools, we have shown critical role of Panx1 channel in neuronal membrane permeation and oxidative injury via activation of phagocytic NADPH oxidase. We have also established a key role of Panx1 in inflammasome activation and interleukin-1 production by retinal ganglion cells.
This is new and exciting project at the Shestopalov Lab that aims to catalogue true microbial diversity at the healthy ocular surface and in disease. It was started in 2009 as a collaborative effort with the BPEI Ocular Microbiology Lab (Dr. D. Miller) and Argonne National Laboratory (Argonne, IL). By 2011 the project grew to include BPEI refractive surgeons Drs. T. O’Brien, V. Perez, E. Alfonso, ophthalmologists A. Galor, S. Yoo, P. Oellers, L. Srur, contact lens specialist Dr. W. Winegar and Dr. R. Van Gelder’s Lab at the University of Washington (Seattle, WA). Sequencing and annotation support is provided by microbial ecologist Dr. D. Nelson (Indiana University) and bioinformaticist Dr. Q. Dong (University of North Texas).Humans body is colonized by thousands of different microbial species, many of which have never been isolated and studied in culture. As a result, our “metagenome” is a composite of Homo sapiens genes and genes contributed by the “microbiome”, consisting of the genomes of the approximately 100 trillion microbes that outnumber human cells ten-to-one. Till recently, healthy ocular surface was considered bacteria-free, since ocular surface microenvironment has potent bactericidal barrier properties. Cornea is, indeed, culture-negative, while the conjunctiva has been shown to contain commensal bacteria. Unexpectedly, our pilot study utilizing deep sequencing of 16S gene amplicon libraries unexpectedly detected hundreds of bacterial species on healthy human conjunctiva (Dong Q, et.al. (2011) IOVS. 52:5408-13) and cornea (in progress). These findings are in good agreement with Dr. VanGelder results on contact lens and conjunctival microbiota, obtained using alternative deep sequencing strategy, Biome Representational in Silico Karyotyping (BRiSK, Muthappan et.al. (2011) Genome Res, 21:626). Such diversity on the healthy ocular surface implies that well-tolerated commensal bacteria are indigenous to the ocular surface homeostasis. True diversity and microbial species prevalens across population groups is yet to be investigated. A pilot project characterizing homeostatic ocular surface microbiota across different genders and age groups has been funded by ARRA R21 exploratory award from NEI in 2010. Preliminary results of this project are being deposited for public access at http://www.microbiota.org/cgi-bin/ocular/20101213/analysis.cgi. Currently, we expanding our quest to study the role of indigenous microbiota in pathogenesis of bacterial keratitis associated with contact lens wear, Acanthamoeba keratitis and dry eye diseases. A new project in collaboration with Dr. W. Winegar (BPEI) will investigate bacterial colonization of contact lens in humans.